1. Field of the Invention
This invention relates to an objective lens used in a multiphoton microscope.
2. Description of Related Art
In order to carry out a three-dimensional observation without breaking a deep part in a living tissue that has been difficult in the use of a conventional confocal laser scanning microscope, special attention has recently been devoted to a microscope using a multiphoton excitation technique (a multiphoton microscope).
The multiphoton excitation technique is the one that excitation generally caused by one photon (a single photon) is done by multiphoton. For example, in a two-photon excitation technique, fluorescence excitation done at a wavelength of 400 nm in a single-photon excitation technique is done at a wavelength of 800 nm twice as great. In this case, the energy of one photon at a wavelength of 800 nm is one-half of that of one photon at a wavelength of 400 nm, and therefore, in the case of a wavelength of 800 nm, two photons are used to cause fluorescence excitation.
In an ordinary single-photon excitation technique, when a substance absorbs light to produce fluorescence, fluorescent energy is less than absorption energy. Consequently, fluorescent light is shifted to the longer-wavelength side than excitation light. For example, serotonin is excited at a wavelength of 260 nm to thereby produce auto-fluorescence with wavelengths of 300–380 nm.
In contrast to this, the multiphoton excitation technique is to detect fluorescent light on the shorter-wavelength side than excitation light by using a wavelength at least twice that of the single-photon excitation technique. For example, in a three-photon excitation technique, serotonin is excited at a wavelength of 750 nm to thereby produce auto-fluorescence with wavelengths of 300–380 nm.
In such a multiphoton excitation technique, it becomes possible to observe fluorescent light with desired wavelengths in an ultraviolet-to-visible region by irradiation of near-infrared laser light.
In the multiphoton microscope, femtosecond ultrashort-pulse laser light with a repetition frequency of about 100 MHz is used as the excitation light in order to cause excitation with a wavelength at least twice greater than the case of one-photon absorption when fluorescent light with identical wavelengths is produced.
The multiphoton microscope is constructed so that, for example, near-infrared ultrashort-pulse laser light emitted from a laser device is collected on a specimen through an objective lens, and fluorescent light produced by multiphoton absorption is detected by a detector through the objective lens, a dichroic mirror, and a collector lens to form a three-dimensional image through a monitor.
According to a two-photon microscope, two-photon absorption is produced in the probability that is proportional to the square of the intensity of excitation light. Additionally, only in an extremely small area close to the focal point of the excitation light, two-photon excitation is caused in the probability that is proportional to the square of a photon density per unit hour and unit volume, and absorption, the production of fluorescence, and a photochemical reaction are localized in the focal spot of a convergent excitation beam, with the result that high spatial resolution is obtained.
According to the multiphoton microscope, fluorescence is produced in only an extremely small part of a specimen, and hence there is the advantage of being able to obtain an image that is very small in depth of focus, such as a so-called tomographic image, even in a thick specimen.
According to the multiphoton microscope, since when fluorescent light with identical wavelengths is produced, excitation is caused with a wavelength at least twice greater than the case of one-photon absorption, the scattering of the fluorescent light in the specimen is reduced by at least one figure and the S/N ratio of fluorescence measurement is improved.
In the multiphoton microscope, as mentioned above, transition energy in the visible region can be excited by the near-infrared pulse light, and excitation light can be transmitted to a considerable depth of a living specimen without undergoing absorption caused by electronic transition or molecular vibration, outside the focal point. Moreover, in the multiphoton microscope, a diffraction-limit microscopic observation image in the ultraviolet region can be obtained by pulse light in the visible region.
In order to generate multiphoton fluorescence, as described above, an extremely high intensity of light is required. However, when multiphoton excitation is caused by a pulse laser of the order of usually used nanoseconds or 100 picoseconds, for example, a living specimen, such as cells, suffers damage because absorption energy is too high. In contrast to this, when the multiphoton excitation is caused by a pulse laser of the order of femtoseconds, a photon density sufficient for the two-photon absorption is provided and the absorption energy can be reduced.
The two-photon excitation, as mentioned above, is caused in the probability that is roughly proportional to the square (the third power in three-photon excitation). of a photon density per unit hour or unit volume. However, when the multiphoton excitation is caused by the pulse laser of the order of femtoseconds, the probability that a plurality of photons exist becomes high because a pulse width is very narrow.
Thus, in the multiphoton microscope, a laser that has an ultrashort pulse of the order of 100 femtoseconds, a near-infrared wavelength, and a repetition frequency of about 100 MHz is used as the most suitable one.
However, laser light emitted from a pulse laser source has some wavelength band, and the pulse width of the laser light is spread each time it passes through an optical element. If many optical elements are arranged and the laser light, after passing through the elements, reaches the specimen, the problem will arise that the pulse width is considerably spread at the position of the specimen and the multiphoton excitation ceases to occur.
As an approach for solving the problem of the spread of the pulse width, a technique (prechirp compensation) that prisms are used to “transmit light with short wavelength ahead of light with long wavelength”, namely to “transmit light with long wavelength behind light with short wavelength”, is generally known. This technique is detailed in “Femtosecond pulse width in microscopy by two-photon absorption autocorrelation”/G. J. Brakenhoff, M. Muller, and J. Squier/J. of Microscopy, Vol. 179, Pt. 3, September 1995, pp. 253–260.
In this technique, a plurality of prisms are arranged along the optical path of a laser beam, which is passed through the prisms to thereby widen the wavelength band, and spacing between the prisms or the relative positions of the prisms themselves to the optical axis are changed so that the optical path length on the long-wavelength side is different from that on the short-wavelength side. Consequently, light with short wavelength is transmitted ahead of light with long wavelength and light with long wavelength is transmitted behind light with short wavelength.
On the other hand, for example, Japanese Patent Kokai No. Hei 06-331898 discloses a conventional objective lens used in a microscope for observation due to single-photon excitation, employing a diffractive optical element as an objective lens which has a high magnification and a high NA and is corrected for aberrations, notably chromatic aberration, without randomly using cemented lens components and anomalous dispersion glass.
Further, for example, Japanese Patent Kokai No. Hei 06-281864 discloses a conventional objective lens used in a microscope for observation due to two-photon excitation.